Epigenetic changes, including DNA methylation, are a common finding in human malignancies. Research in chronic lymphocytic leukemia (CLL) has focused on genetic deletions that promote impaired apoptosis, but to date very few genes have been identified. In preliminary studies we have utilized Restriction Landmark Genomic Scanning (RLGS), a two-dimensional gel electrophoresis that allows detection of altered DNA methylation patterns, to study methylation in ten CLL patient samples. Our results strongly indicate that DNA methylation, via gene silencing, contributes significantly to the pathogenesis of CLL. Specifically, we demonstrated marked variation in the amount of aberrant methylation in patient samples ranging from 2.5-8.1% as compared to normal B cells. To fully establish the role of methylation in CLL and to exploit these alterations in the clinic, we propose more detailed studies outlined below. Our hypothesis for this Project is that DNA methylation contributes significantly to the development and progression of CLL. In Specific Aim 1, RLGS will be used on 100 CLL patients of different genotypes. RLGS profiles of CLL patients samples will be compared with CD19+ selected normal B-lymphocytes to identify novel CLL methylation sequences specific to CLL. In addition, methylation patterns between favorable and un-favorable genotypes will be compared to identify the most promising gene targets to pursue for investigation in Specific Aim 2. In Specific Aim 2, we plan to clone as many as 200 novel methylated sequences identified from aim 1 using genomic libraries that will guide the search for new tumor suppressor genes in CLL. Furthermore we propose experiments to perform a biostatistical evaluation of DNA methylation patterns in CLL. In aim 3 these genes will then be analyzed in a large cohort of CLL patient samples obtained from the national cooperative oncology groups, and results will be correlated with response to therapy, progression-free survival and overall survival. In Specific Aim 4, novel candidate cancer genes, DAPK1, ID4 and DERMO1, whose expression is altered by DNA methylation, will be studied in more detail in order to determine potential importance to the pathogenesis and progression of CLL.